microRNAs (miRNAs) are sequence-specific regulators of gene expression that impact almost all biological processes in diverse eukaryotes. Defects in miRNA levels or activities are associated with numerous diseases. Both biogenesis and degradation contribute to the steady-state levels of miRNAs in vivo. The basic molecular framework underlying miRNA biogenesis has been elucidated. In contrast, although studies in ciliate, algal, plant, and animal models have implicated the existence of conserved processes that degrade miRNAs and related small RNAs, such as small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), the enzymes that degrade small RNAs have yet to be identified in most organisms. As such, a basic framework of miRNA degradation awaits further studies. The goal of this project is to establish such a framework. The project capitalizes on recent advances in the area of miRNA degradation made in the PI's laboratory using the Arabidopsis model. The PI's lab identified the enzymes responsible for two conserved miRNA degradation processes in eukaryotes, 3' truncation and 3' uridylation (addition of a short, U-rich tail to miRNAs). The proposed research employs a combination of genetics, genomics, and biochemical approaches to examine the activities, interdependence and concerted actions of these enzymes with the goal of establishing a general framework of miRNA degradation. The PI's lab has also gathered preliminary evidence that implicates endogenous target mimic RNAs in miRNA turnover. The project will examine how the interplay between target mimic RNAs and the general miRNA degradation machinery results in the turnover of specific miRNAs. By elucidating principles governing miRNA degradation, the project will generate far-reaching impacts. As mounting evidence points to conserved molecular mechanisms underlying miRNA degradation in diverse eukaryotes, the proposed studies using the Arabidopsis model, from which two conserved miRNA degradation processes were first described and the enzymes responsible for these processes were first identified, will establish a general framework of miRNA degradation that is likely applicable to other eukaryotes including humans. The knowledge will enrich our understanding of various biological processes that are influenced by miRNAs and enhance our ability to control miRNA abundance to treat diseases. The molecular framework of miRNA degradation is also likely applicable to siRNAs or piRNAs, which are emerging as agents that confer epigenetic memory in plants and animals, thus further broadening the impacts of the work.